Substance P (RPKPQQFFGLM-NH2) [M + 3H](3+) ions have been shown to occupy two distinct conformer states, a compact population of conformers that is formed by evaporation of hydrated ions, and an elongated population of conformers that is formed by collisional heating of the compact conformer. Molecular dynamics (MD) simulations and amino acid mutations revealed that the compact conformer is stabilized by intramolecular interactions between the localized charge-carrying sites, specifically the N-terminus, R(1), and K(3), with the side chains of glutamine and phenylalanine residues present in the peptide. Here, we employ amino acid mutations and cryogenic ion mobility-mass spectrometry (cryo-IM-MS) in an effort to understand how eliminating specific intramolecular interactions alters ion hydration, as well as the dehydration dynamics of substance P during the final stages of the electrospray process. The results clearly illustrate a direct link between the stabilizing effects of intramolecular self-solvation and the formation of substance P [M + 3H](3+) ions. Most notably, removal of these stabilizing interactions leads to a reduction in the abundances of [M + 3H](3+) ions induced by charge reduction reactions, i.e., loss of H+(H2O)(n) ions to form [M + 2H](2+) ions during the final stages of the electrospray process.